Bacterial chemotaxis, the ability of prokaryotes to adapt their motion to external stimuli, has long stood as a model system for understanding transmembrane signal transduction, intracellular information propagation, and motility. Furthermore, many human pathogens such Vibrio cholerae, Helicobacter pylori, Treponema pallidum (syphilis) and Borrelia burgderfori (lyme disease) rely on chemotaxis and motility to establish infection. The signaling network underlying chemotaxis displays amazing sensitivity, robustness, dynamic range and even a rudimentary molecular memory. The goal of this project is to understand in molecular detail how bacterial cell surface receptors interact with and modulate the activity of the central chemotaxis histidine kinase CheA. CheA, in response to receptor input, phosphorylates the second messenger CheY, which in its activated form directly modulates the rotation of the flagellar motor. Receptors (also known as MCPs), CheA and the coupling protein CheW form large assemblies in the cytoplasmic membrane (the "chemosome") where their cooperative interactions manifest the response properties of the system. To probe the architecture of the chemosome a combination of biophysical techniques centered on X-ray crystallographic structure determination and pulsed dipolar electron-spin resonance spectroscopy (PDS) of spin-labeled proteins will be applied. Subjects of study include reconstituted soluble complexes of CheA, CheW and MCP domains, as well as full-length chemoreceptors solubilized in detergents and incorporated into nanodiscs. Residue substitutions that mimic receptor modification will be used to set complexes and receptors in different states of activity. Emphasis will be placed on the chemotaxis systems from non-enteric bacteria, which include many human pathogens and present interesting and important differences compared to the E. coli chemotaxis system. In addition to ligand-binding MCPs, two other types of CheA-interacting receptors will be investigated: 1) naturally soluble receptors that do not contain transmembrane regions and 2) "energy-sensing" receptors such as E. coli Aer that report the redox state to CheA. Key issues to be addressed include: how receptors interact with the kinase in states of both activation and inhibition;how ligand binding and receptor modification tunes kinase activity;the role of CheW in coupling the receptors to the kinase;and the function of higher order assemblies in generating the hallmark high gain, sensitivity and dynamic range of bacterial sensory responses. Mechanisms of signal termination and receptor site modification will also be investigated by structural means. Insights gained from biophysical studies will be fed into cell-based experiments to test functional relevance. Given that bacterial chemotaxis proteins do not have close mammalian homologs, these systems offer promising targets for the design of antimicrobial agents. PUBLIC HEALTH RELEVANCE Due to its relative simplicity, and well-established experimental systems, bacterial chemotaxis provides perhaps the best opportunity for understanding how surface receptors send signals across cell membranes. Moreover, many classes of bacterial pathogens including Vibrio cholerae (cholera), Helicobacter pylori (ulcers and gastric cancer), Treponema pallidum (syphilis) and Borrelia burgderfori (lyme disease) rely on chemotaxis and motility to invade tissues and evade the immune system. Because the components that compose the underlying signaling networks are largely orthogonal from those found in mammals, they provide excellent targets for the development of antimicrobial agents.